Photoprotected Peptide α‐Ketoacids and Hydroxylamines for Iterative and One‐Pot KAHA Ligations: Synthesis of NEDD8

Abstract

The convergent synthesis of proteins by multiple ligations requires segments protected at the N-and/or C-terminus with masking groups that are orthogonal to the acid-and base-labile protecting groups used in Fmoc-SPPS. They must be stable to solid-phase peptide synthesis, HPLC purification, and ligation conditions and easily removed in the presence of unprotected side chains. In this report, we document photolabile protecting groups for both a-ketoacids and hydroxylamines, the key functional groups employed in the a-ketoacid-hydroxylamine (KAHA) ligation. The novel photoprotected a-ketoacid is easily installed onto numerous different C-terminal peptide aketoacids and removed by UV light under aqueous conditions. These advances were applied to the one-pot synthesis of NEDD8, an important modifier protein, by three different convergent routes. These new protecting groups provide greater flexibility on the order of fragment assembly and reduce the number of reaction and purification steps needed for protein synthesis with the KAHA ligation.Scheme 1. Protection of a-ketoacids as photolabile cyclic acetals.Helv. Chim. Acta 2016, 99, 868 -894 In order to utilize the photoprotected a-ketoacids for iterative segment ligations, we applied them to the Fmoc-SPPS of C-terminal protected peptides. Using the same linker we previously reported for traceless preparation of C-terminal peptide a-ketoacids, we Scheme 2. Synthesis of enantiomerically enriched protecting group 2 and determination of the absolute configuration of acetal derivative 7 by single-crystal X-ray diffraction (thermal ellipsoids shown in the ORTEP model are set at a 50% probability level). 870Helv. Chim. Acta 2016, 99, 868 -894 www.helv.wiley.com a Determined by chiral SFC (column Chiralpak ADH) with detection at 220 nm. b After recrystallization from hexanes/AcOEt 25:1 (74% recrystallization yield). CatBH = catecholborane.Scheme 5. Synthesis of photoprotected (S)-5-oxaproline 21.Helv. Chim. Acta 2016, 99, 868 -894 873 Scheme 6. One-pot synthesis of NEDD8 in the N-to C-direction using a photoprotected tyrosine a-ketoacid.Helv. Chim. Acta 2016, 99, 868 -894 875Scheme 8. One-pot convergent synthesis of NEDD8 from four segments.Helv. Chim. Acta 2016, 99, 868 -894 877

Chemical Synthesis of the Highly Hydrophobic Antiviral Membrane‐Associated Protein IFITM3 and Modified Variants

“…[20][21][22][23] We decided to employ an oligo-arginine tag (ArgTAG)s imilar to one described by Wade and co-workers, [24] which can be removed by basic treatment at room temperature.T his tag presents two advantages:f irst, oligoarginines have been shown to greatly increase the solubility of hydrophobic sequences,t hus making the handling and purification of these segments easier.S econd, this arginine tag is attached to the Cterminus rather than to the backbone of the peptide,thus facilitating its synthesis and incorporation into the peptide.W ith this strategy,F moc-SPPS of the hydrophobic segment proceeded smoothly,a nd the cleaved peptide bearing the (Arg) 7 tag could be readily purified by reverse-phase HPLC.T he synthesis on one gram of resin afforded 128 mg of the purified peptide 3 (7 %overall yield) bearing an N-terminal 5-oxaproline.T he central segment, bearing aC -terminal valine a-ketoacid, was prepared by Fmoc-SPPS,a nd the requisite photoprotected [(2-nitrophe-nyl)ethyl]carbamate-Opr (NpecOpr) was introduced as the final residue. [25] Thefragments NpecOpr-S2-KA (2)and Opr-S3-ArgTAG (3)were joined by the first KAHA ligation. We were pleased to find that the segments were perfectly soluble in a9 5:5 NMP/H 2 Omixture at 20 mm.Despite the use of the hindered valine a-ketoacid, the ligation was almost complete after 10 h, by which time ag el had formed.…”

Facile Synthesis of Internal and C‐Terminal Peptide α‐Ketoamides with Fmoc‐Solid Phase Peptide Synthesis

“…We have recently introduced α ‐ketoacids orthogonally protected with a photolabile protecting group . By using these same starting materials, we successfully prepared a peptide bearing an internal photocaged α ‐ketoamide.…”

Chemoselective Derivatization of Folded Synthetic Insulin Variants with Potassium Acyltrifluoroborates (KATs)

“…We also found the unprotected form to be somewhat unstable to basic buffers and lyophilization. Encouraged by success with other photoprotected hydroxylamines and α-ketoacids for peptide assembly by KAHA ligation, [20] we designed a photoprotected variant of the Fmoc-ornithine hydroxylamine (4) (Scheme 2,a). This building block would stay protected during SPPS and TFA cleavage, allowing the assembly of longer peptides by KAHA ligation and further synthetic steps, such as Acmdeprotection and protein folding.…”